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Abstract

Background

Iodine-123-β-CIT, a single-photon emission computed tomography (SPECT) ligand for
dopamine transporters (DATs), has been used for in vivo studies in humans, monkeys, and rats but has not yet been used extensively in mice.
To validate the imaging and analysis methods for preclinical DAT imaging, wild-type
healthy mice were scanned using 123I-β-CIT.

Methods

The pharmacokinetics and reliability of 123I-β-CIT in mice (n = 8) were studied with a multipinhole SPECT/CT camera after intravenous injection
of 123I-β-CIT (38 ± 3 MBq). Kinetic imaging of three mice was continued for 7 h postinjection
to obtain the time-activity curves in the striatum and cerebellum volumes. Five mice
had repeated measures 4 h post-123I-β-CIT injection to provide an indication of test-retest reliability. The same five
mice served as a basis for a healthy mean SPECT template.

Results

Specific binding of 123I-β-CIT within the mouse striatum could be clearly visualized with SPECT. The kinetics
of 123I-β-CIT was similar to that in previously published autoradiography studies. Binding
potential mean values of the test-retest studies were 6.6 ± 15.7% and 6.6 ± 4.6%,
respectively, and the variability was 9%. The SPECT template was aggregated from the
first and second imaging of the test-retest animals. No significant difference between
the templates (P > 0.05) was found. From the test template, a striatal volume of 22.3 mm3 was defined.

Conclusions

This study demonstrates that high-resolution SPECT/CT is capable of accurate, repeatable,
and semiquantitative measurement of 123I-β-CIT DAT binding in the mouse brain. This methodology will enable further studies
on DAT density and neuroprotective properties of drugs in mice.

Keywords:

SPECT/CT; Dopamine transporters; 123I β-CIT; Tracer kinetics

Background

Previously, sensitivity and resolution of the single-photon emission computed tomography
(SPECT) system have been challenges in small-animal imaging. Currently, the scanning
time can be kept short enough to enable dynamic acquisitions, due to the high level
of detection efficiency and excellent spatial resolution obtained by multipinholes
[1]. Here, we examined the feasibility of dual-modality-dedicated small-animal SPECT/computed
tomography (CT) in submillimeter brain structures and the advantage of dynamic acquisitions.

Parkinson's disease (PD) is characterized by the progressive degeneration of nigrostriatal
dopaminergic neurons. This neurodegenerative process is associated with a loss of
striatal dopamine transporters (DATs)
[2,3]. In vivo measurement of DAT density with SPECT is an early marker of dopaminergic cell loss
in subjects with PD
[4-6]. 123I-2β-carbomethoxy-3β-(4-iodophenyl)tropane (123I-β-CIT), a SPECT ligand for DATs, has not yet been validated in mice, which are frequently
induced to model several diseases. Previous mouse studies are ex vivo autoradiography works
[7-9], in which follow-up of the same animals was not possible and test-retest measurement
was disabled. Whereas, Mochizuki et al. used a simple probe system offering only a
single projection and therefore cannot differentiate between the brain structures
[10]. However, the full benefit of small-animal imaging can only be achieved by in vivo quantification of radiotracers that enable the following of physiological processes
over time in the same animal. In rats, such quantification has been performed using
clinical gamma cameras
[11,12]. The latest work was executed with a modern small-animal multipinhole SPECT by taking
static images of the mouse brain
[13]. In that work, the used DAT tracer was 123I-2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl)nortropane (FP-CIT; DatScan, GE Healthcare Inc., Waukesha, WI, USA),
which is divergent from 123I-β-CIT, as will be discussed later in this chapter. The aim of our study was to determine
the complimentary preclinical results since the kinetics between mice and rats may
vary.

Absolute quantification of DATs in mice would require full kinetic modeling, with
invasive arterial blood sampling and dynamic SPECT scanning. However, arterial blood
sampling in mice is difficult, and other methods have been accepted for kinetic analysis
[14,15]. For 123I-labeled tracers, good correlation has been demonstrated using the semiquantitative
index of the specific DAT binding, referred to as binding potential (BP). BP is a
ratio of the specific to nonspecific binding of DATs (such as the striatum and cerebellum).
BP is directly proportional to the density of DATs in the equilibrium state
[14,15]. We used BP to analyze the 123I-β-CIT ratios in the kinetics and test-retest studies of mice.

The 123I-β-CIT and FP-CIT are both clinically available and specific for use in PD diagnostics
[16]. Here, we used 123I-β-CIT for several reasons: First, more rapid wash-up of the FP-CIT may cause underestimation
of DAT density in patients
[17]. Second, more rapid kinetics of FP-CIT in patients is even further accelerated in
mice, which makes 123I-β-CIT more suitable for investigating disease models or pharmaceutical effects in
mice. Third, with 123I-β-CIT, higher binding ratios have been reported in humans
[17], which improve the signal-to-noise ratio in images acquired from the mouse brain.

To demonstrate the feasibility of the DAT tracer 123I-β-CIT in preclinical research in mice, we established the kinetics of the tracer
with high-resolution SPECT/CT imaging. Furthermore, we validated the steady-state
conditions of the tracer in the brain and evaluated the reproducibility of the method
in test-retest measurements. Our second objective was to create a normal template
to be used as a future reference frame for the coregistration and analysis of the
mice with altered DAT production.

Methods

SPECT/CT system

SPECT/CT imaging was performed using a preclinical four-headed gamma camera with integrated
CT system and dedicated multipinhole collimators, comprising in all 36 1.0-mm pinholes
(nanoSPECT/CT, Bioscan Inc., Washington, DC, USA). The manufacturer states that the
sensitivities of the pinhole collimators are >1,200 counts per second (cps)/MBq, with
a maximum resolution of ≤0.75 mm. The scanning mode is helical for both modalities.

Phantom studies

The spatial resolution of the system was verified for 123I, using a Jaszczak phantom with hot rods ranging from 0.7 to 1.2 mm (Bioscan Inc.,
Washington, D.C., USA). The phantom was filled with 75 MBq of 123I-NaI (MAP Medical Technologies Oy, Tikkakoski, Finland), and the images were acquired
in 32 projections, using 150 s per gantry position. The imaged data were reconstructed
with HiSPECT NG software (Scivis GmbH, Göttingen, Germany).

Animals

The animal experiments were reviewed and approved by the Finnish National Animal Experiment
Board and performed in accordance with Good Laboratory Practices for Animal Research.
Triple-mixed genetic background 129Ola/C57BL/6/ICR mice were backcrossed once to the
129Ola line, and 4-month-old male progenies were used in the SPECT experiments. The
animals were maintained under isoflurane inhalation anesthesia during radiotracer
administration and imaging. Under anesthesia, body temperature of the animals was
maintained using a heated animal bed (37°C) (Minerve, France), during imaging, and
with a heating pad under the cage, between imaging sessions.

At the early time points (from 20 min to 2 h 30 min) of the kinetics experiments (n = 3), the data were acquired dynamically with a scanning length of 15 min (angular
step 15°, 24 projections, 150 s per gantry position). At the late time points (at
4 h and 7 h), the scanning length was 25 min (angular step 15°, 24 projections, 250
s per gantry position). In all, 11 time points were collected from the mice. The data
were acquired in a 128 × 128 matrix with pixel size and a slice thickness of 0.2 mm.

CT was acquired at 20 min and later at 4 and 7 h. The CT parameters used in the present
work were as follows: 180 projections, pixel size of 192 × 192 μm, X-ray source of
45 kVp, and exposure time of 500 ms. Helical scanning is used by both modalities and
is performed by moving the animal through the SPECT and CT.

In the test-retest experiment (n = 5), the mice were imaged 4 h postinjection, which was confirmed to be the equilibrium
time point in the kinetics study. The same animals went through the same protocol
after 8 days. The same SPECT protocol was used as described for the late time points
in the kinetics experiment.

Data analysis: kinetics experiment

The data were reconstructed in the system's dedicated reconstruction program with
an iterative reconstruction algorithm, resulting in a voxel size of 0.3 mm. After
image reconstruction, the images were straightened and analyzed using InVivoScope
software (Bioscan Inc.). Straightening of the images was assisted by the CT images.
To avoid the variability of the slice selection and to gain statistical power, we
used the entire striatum volume for the analysis. Time-activity curves were obtained
from the kinetics data by manually delineating the volumes of interest (VOIs). At
each time point, the VOIs were drawn over specific (striatal) and nonspecific (cerebellar)
brain structures, and the mean counts in these two areas were measured against time.
From the mean values obtained, we calculated the BP, which represents the ratio of
the distribution volumes of the specifically and the nonspecifically bound compartment.

Data analysis: test-retest experiment

Following data reconstruction, the images were first manually straightened and converted
from Dicom to the Analyze format
[18] and further processed in Vinci (S. Vollmar et al., Cologne, Germany)
[19]. In Vinci, the images were first masked to remove the eyes and other possible high-uptake
areas external to the brain. Then, all the images were normalized individually using
the average value of the voxels within 95% of the maximum count value in the striatal
volume. Finally, both time points were registered to the SPECT template (see below),
which served as a reference space and defined the VOIs. The mean count density per
pixel in each region was calculated and corrected for the effects of decay. The test-retest
variability was calculated as the absolute difference of two measurements divided
by the mean of two measurements as a percentage. Comparison between the test and retest
time points was achieved by the Wilcoxon signed rank test.

Creation of template

Two separate templates were aggregated from the test and retest mice groups. The images
were first manually straightened, masked, and normalized to 95% of the maximum count
value. Secondly, the images were spatially coregistered using an affine transformation
algorithm with mutual information as a similarity measure for registrations. Then,
the average and standard deviation templates were calculated using the spatial- and
count-normalized images. Finally, the anatomical areas were confirmed by coregistering
the SPECT template to an available magnetic resonance imaging (MRI) brain template
[20]. Comparison between the test and retest templates was achieved by voxel-by-voxel
t test.

Results

SPECT imaging of phantom

Prior to the animal studies, we evaluated the performance of the scanner for spatial
resolution. Under the conditions of high radioactivity (75 MBq), acquisition time
(20 min), and reconstruction parameters for fine image quality, the resolution was
superior. Figure
1 shows the SPECT images and their count profile curves of the Jaszczak phantom filled
with 123I-Na, yielding an excellent spatial resolution with good visualization of hot rods
as thin as 0.8 mm.

Figure 1.Jaszczak phantom. The spatial resolution of the system was verified for 123I, using a Jaszczak phantom with hot rods ranging from 0.7 to 1.2 mm. Images were
acquired in 32 projections, using 150 s per gantry position. Jaszczak phantom, filled
with 75 MBq of 123I-Na, demonstrates the spatial resolution of the system. The red line in the SPECT
image (a) represents the area where the count profile curve (b) is captured. The 0.8-mm hot rods are clearly detectable.

SPECT template

Templates were compared using voxel-by-voxel t test, and no significant differences were found between test and retest. Figure
2 shows the outcome of coregistration between the created SPECT template and MRI template.
The anatomical areas seem to be equivalent between SPECT and MRI (Figure
2).

Figure 2.Coregistered template. In building the template, the anatomical areas are confirmed by coregistering the
template to an available MRI brain template
[20]. Coregistration is visualized so that SPECT is in color and MRI is in gray scale.

In the individual animals and the template, the anatomical volume of the striatum
was estimated by several thresholds from the maximum intensity of the structure (Figure
3). From the template, we obtained a striatal volume of approximately 22.3 mm3 by choosing a threshold of 50% of the maximum intensity of the striatum (Figure
3). When performing the same analysis for individual animals, we obtained a striatal
volume of 17.2 mm3 ± 20% (Figure
3).

Figure 3.Striatal volumes. Mice (n = 5) received 1.12 ± 0.08 MBq/g b.wt. of 123I-β-CIT. They were imaged 4 h postinjection, and scanning length was 25 min (angular
step 15°, 24 projections, 250 s per gantry position). The data were acquired in a
128 × 128 matrix with pixel size and a slice thickness of 0.2 mm. The anatomical volume
of the striatum was estimated by several thresholds from the maximum intensity of
the structure. A striatal volume was measured for the template and individual mice
by choosing a threshold of 50% of the maximum intensity of the striatum (vertical
line), which resulted as a striatal volume of 22.3 mm3 and 17.2 mm3 ± 20%, respectively.

Dopaminergic terminals are present at high density in the striatum but are less dense
in the cerebellum, which makes delineation of the nonspecific uptake area very difficult.
Thus, the cerebellar VOI was chosen to consist of only part of the cerebellum (13.3
mm3) to avoid delineation of those areas that consist of counts due to scattered or partial
volume effects. Meanwhile, the cerebellar volume should be sufficiently large to gain
enough statistics for calculation of a reliable BP.

Kinetics study of mice

In the kinetics experiment, a total of 11 time points were imaged, starting at 20
min and ending at 7 h postinjection (Figure
4a). In the striatum, the amount of activity increased gradually and peaked at approximately
2 to 3 h (Figure
4b). After 4 h, the striatum uptake washed out gradually. The nonspecific binding area,
the cerebellum, showed no binding and half of the activity dissipated in 2.5 h. The
ratio of the specific binding value of 123I-β-CIT to nonspecific uptake, BP, became constant 2 h postinjection (Figure
4c). After 4 h, BP shows a gradual increase. Symmetrical striatal binding was visible
in the SPECT images at 4 h postinjection (Figure
4a). Harder's glands showed radioactivity uptake (Figure
4a).

Figure 4.Kinetics experiment data. Mice (n = 8) received 34 to 41 MBq of 123I-β-CIT. At the early time points (from 20 min to 2 h 30 min) post-tracer injection,
the data were acquired dynamically with a scanning length of 15 min (angular step
15°, 24 projections, 150 s per gantry position). At the late time points (at 4 h and
7 h), the scanning length was 25 min (angular step 15°, 24 projections, 250 s per
gantry position). The data were acquired in a 128 × 128 matrix with pixel size and
a slice thickness of 0.2 mm. In all, 11 time points were imaged (a). Time-activity curves were obtained from the kinetic data by manually delineating
the VOI (b). At each time point, the VOIs were drawn over specific (striatal) and nonspecific
(cerebellar) brain structures, and the mean counts in these two areas were measured
against time. Specific-to-nonspecific BP was calculated (c).

Test-retest study

In the test-retest study, the 4-h time point was set as the acquisition time, based
on the kinetics experiment. The same animals followed the same protocol after 8 days.
The individual BP values obtained from each mouse are shown in Figure
5. The mean BP values of the test-retest studies were 6.6 ± 15.7% and 6.6 ± 4.6% (mean
± relative standard error), respectively, and these measures did not differ statistically
from each other (P = 0.968). The test-retest variability was 9%.

Figure 5.Binding potential values. The test and retest mice (n = 5) received 1.12 ± 0.08 MBq/g b.wt. and 0.94 ± 0.07 MBq/g b.wt. of 123I-β-CIT, respectively. Mice were imaged with the equivalent protocol in the test and
retest time points; scanning length was 25 min (angular step 15°, 24 projections,
250 s per gantry position). The data were acquired in a 128 × 128 matrix with pixel
size and a slice thickness of 0.2 mm. Individual BP values were obtained at 4 h postinjection
on each of the five mice at both test and retest (8 days later), with the corresponding
mean and standard deviation. The test-retest measurements were insignificantly different
(P = 0.968).

Discussion

The primary aim of this study was to demonstrate that small-animal SPECT/CT imaging
is capable of measuring DAT density. We validated a DAT-binding imaging protocol for
the mouse brain, using 123I-β-CIT. To our knowledge, the present study is the first to demonstrate the 3D kinetics
of 123I-β-CIT in mice, using SPECT/CT imaging.

The SPECT/CT used was equipped with four collimators and apertures, each with nine
1-mm pinholes, resulting in high counting rates. The resolution for the camera was
in the submillimeter range; Jaszczak phantom imaging showed that we can reliably image
the mouse striatum.

From the template, we obtained a striatal volume of approximately 22.3 mm3 with a threshold of 50% of the maximum intensity of the striatum. Similar striatal
volumes have been reported in equivalent strains
[21]. At the same threshold, we obtained a striatal volume of 17.2 mm3 ± 20%. In Figure
3, the striatal volume is shown as a function of thresholds in individuals and the
template. The striatal volume increases until it becomes stable at approximately 50%
to 60% of the maximum activity. The phenomenon was found in both cases (Figure
3). For threshold values higher than 60%, the volume spreads outside the striatal volumes,
into other parts of the brain. The higher striatal volume in the template is due to
individual variation, coregistration, and averaging. This comparison indicates that
the template represents data in healthy mice well and that no coregistration or technical
problems occurred. Further, we compared the templates created from the first and second
imaging of the test-retest study and found no significant difference. This result
demonstrates that our method has good repeatability. In future works, we will address
individual variation in striatal structures and minimize the processing steps needed
using the template.

In the present study, we report the kinetics of the 123I-β-CIT tracer in the striatum and cerebellum. As indicated earlier, the quantitative
validity of the BP method (striatal to background brain region ratio) depends on the
achievement of steady levels of activity in these regions
[17]. As shown in Figure
4b, 3 h postinjection, there is parallel washout in cerebellum and striatal volumes.
Thus, BP analysis can be considered valid 4 h post-123I-β-CIT injection in the mouse brain. These results agree with the earlier autoradiography
works
[7-9]. However, our results show moderate increase in BP, which is evident at the 7-h imaging
point. This might be due to the individual kinetics of mice and also because of low
statistics in the cerebellum volume. To resolve this, further kinetic studies should
be performed at late imaging points.

We used BP to perform semiquantitative analysis of dopaminergic neurotransmission
post-123I-β-CIT injection. BP increased over the time period followed, similar to previous
rat studies with SPECT
[11,12] and autoradiography work with both rats
[22] and mice
[7-9]. In healthy human subjects, the evolvement of BP is similar, differing only by time
scale
[23,24], due to the slower metabolism of humans compared with mice. In the present work,
4 h postinjection was a suitable time point for use in the test-retest study, which
is equivalent to 20 to 24 h in human patients
[23,24].

In the test-retest study, one of the five animals showed a clearly larger variation
among BPs, which probably resulted from unsuccessful injection at either one of the
time points. The variability between test and retest was clearly better than that
in healthy human subjects
[25,26], which shows that our method has good test-retest reproducibility. Such improvement
was expected, due to the better sensitivity and resolution of the small-animal SPECT
instrument compared with clinical systems.

Successful validation of the imaging protocol and establishment of the template will
help to reduce the number of control mice needed for the experiments and will further
reduce the overall costs and time needed. The template created here will be further
used as a reference to differentiate between normal and genetically modified mouse
DAT densities. Also, we are planning to extend the template to include serotonin transporters.
In present and future works, the template will offer a reference for coregistration
and will take into account the variance between individuals in the VOI analysis. Furthermore,
correct and repeatable delineation of VOIs is needed to gain enough statistical power
because the targets are submillimeter in size. Herein, we focused on certain VOIs,
but the template may also be used in voxel-by-voxel analysis.

Conclusions

We have demonstrated explicitly that high-resolution, multipinhole SPECT/CT of mice
is capable of accurate, repeatable, and semiquantitative measurement of DAT binding,
using 123I-β-CIT. This methodology should increase the opportunities for further study of cerebral
binding sites, especially in mice.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MP, EH, and MR carried out the SPECT/CT experiments, participated in the design of
the study, and drafted the manuscript. MP and EH carried out the data analysis and
template creation. J-OA and MS involved in conceiving the study and provided the animals.
AU, PTM, and SS were involved in conceiving the study and revising the manuscript.
KB conceived the study, participated in its design and coordination, and helped draft
the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The work was supported by Tekes project ‘SPECT/CT in preclinical drug research.’ We
thank Mari Rissanen for technical assistance.